Merging Directed C–H Activations with High-Throughput Experimentation: Development of Iridium-Catalyzed C–H Aminations Applicable to Late-Stage Functionalization

Herein, we report an iridium-catalyzed directed C–H amination methodology developed using a high-throughput experimentation (HTE)-based strategy, applicable for the needs of automated modern drug discovery. The informer library approach for investigating the accessible directing group chemical space, in combination with functional group tolerance screening and substrate scope investigations, allowed for the generation of reaction application guidelines to aid future users. Applicability to late-stage functionalization of complex drugs and natural products, in combination with multiple deprotection protocols leading to the desirable aniline matched pairs, serve to demonstrate the utility of the method for drug discovery. Finally, reaction miniaturization to a nanomolar range highlights the opportunities for more sustainable screening with decreased material consumption.

Reagents were dispensed by acoustic dispensing using the Labcyte Echo® liquid dispensing system. For the reaction set-up Labcyte Echo® qualified 1536-well low dead volume plates were used. As source plates and analytical plates Labcyte Echo® qualified 384-well plates were used. LCMS analysis (MS only, no UV) was carried out on a Waters Acquity UPLC system, on a BEH C18 column (basic method: A: H2O/MeCN/NH3 = 95/5/0.2, B: MeCN). MS analysis was also performed on an AMI-MS system as previously described. Screening and HTE information: Reaction set-up and analysis (5 µmol -0.02 mmol scale) Solids were weighed in manually or using the Quantos powder dosing system from Mettler Toledo. For liquid dispensing manual pipettes and the TECAN freedom evo liquid handling platform was used. For solvent removal the SP Scientific Genevac HT-6 evaporation system was used. The reactions were set-up in a Para-Dox® 24-or 96-position parallel synthesis plate using 50 µL or 1 mL vials. LCMS analysis was carried out on a Waters Acquity UPLC system, on a BEH C18 column (basic method: A: H2O/MeCN/NH3 = 95/5/0.2, B: MeCN).

Screening and HTE information: Purification (0.02 mmol scale)
Pre-purification analysis of the crude samples was performed on a Waters Acquity System with a Waters QDA Mass Spectrometer using three different columns to evaluate the best method for preparative separation: Waters Acquity UPLC HSS C18 1.8 µm (2.1 x 50 mm) and Waters Acquity UPLC CSH Fluoro-Phenyl 1.7 µm (2.1 x 50 mm) at acidic conditions and Waters Acquity UPLC BEH C18 1.7 µm (2.1 x 50 mm) at basic conditions. A gradient of 2-94% MeCN (10 mM formic acid or 0.2% ammonia) was used with a flow 0.8 mL/min for 2.5 min at 45°C.
Preparative LC was performed on a Waters Fraction Lynx system with a Waters QDA Mass Spectrometer and a 2767 autosampler. The columns used were either Waters SunFire C18 OBD 5 µm (10 x 100 mm) or Waters XSelect CSH Fluoro Phenyl OBD 5 µm (10 x 100 mm) at acidic conditions or Waters XBridge BEH C18 OBD 5 µm (10 x 100 mm) at basic conditions. A focused 50 % MeCN gradient (0.1 M formic acid or 0.2% ammonia) was used with a flow of 8.3 ml/min in 5.2 min at ambient temperature. Fraction collection was triggered using a combination of UV and MS.
The purity of the isolated sample was determined using a Waters Acquity System with a Waters QDA Mass Spectrometer at both acidic and basic conditions, using a Waters Acquity UPLC HSS C18 1.8 µm (2.1 x 50 mm) at low pH and Waters Acquity UPLC BEH C18 1.7 µm (2.1 x 50 mm) at high pH. A gradient of 5-94% MeCN (10 mM formic acid or 47 mM NH3/ 6.5 mM NH4HCO3) was used with a flow 0.8 mL/min for 1.5 min at 50°C.

Analytical information
LCMS analysis was carried out on a Waters Acquity UPLC system, on a BEH or HSS C18 column. For SFC-MS analysis a Waters Acquity UPC2 SFC-MS system with a BEH column was used. Conversion quantification in the optimization campaign, informer libraries and functional group tolerance studies were based on UV trace as a ratio of SM:P (starting material : product). Nuclear magnetic resonance spectra ( 1 H, 13 C, 19 F, COSY, HSQC, HMBC) were recorded on Bruker ULTRASHIELD 500 and 600 MHz spectrometer with a Bruker CRYO PLATFORM. 1 H NMR spectra were referenced to CD3OD (3.31 ppm), DMSO-d6 (2.50) and CDCl3 (7.26). 13 C NMR spectra were recorded at 126 and 151 MHz, referenced to in CD3OD (49.00 ppm), DMSO-d6 (39.52) and CDCl3 (77.16 ppm). Coupling constant (J) values were measured in Hertz (Hz) and chemical shift (δ) values in parts per million (ppm). HRMS data was recorded on a Waters Acquity System with a XEVO-QTOF MS spectrometer at either acidic or basic conditions, using Leucine Enkephaline (C28H37N5O7, m/z 556.2771) as lock mass. ESI ionization in positive or negative mode. A gradient of 5-90% MeCN (10 mM formic acid or 47 mM NH3/ 6.5 mM NH4HCO3) was run with a flow of 0.8 mL/min for 2.5 min at 45°C, using a Waters Acquity UPLC HSS C18 1.8 µm (2.1 x 50 mm) at low pH and Waters Acquity UPLC BEH C18 1.7 µm (2.1 x 50 mm) at high pH.

Safety considerations
Organic azides are known high energy compounds and special precautions need to be taken when used. As N2 gas is formed as a side-product in the reaction, measures to mitigate the pressure buildup should be taken. Keeping a 1:3 reaction volume to head space is recommended.

Catalyst preparation
The [Cp*Ir(H2O)3]SO4 catalyst was prepared according to published procedure. 2 Ag2SO4 (2.52 mmol, 786 mg) was added to a suspension of [Cp*IrCl2]2 (1.255 mmol, 1.00 g) in deionized H2O (8.0 mL). The mixture was stirred at room temperature for 16 h. The contents were then transferred into a Corning® 15 mL centrifuge tube and centrifuged at 2000rpm for 1 minute to sediment the silver salts. The supernatant was removed and kept. The cake was washed with water (8 mL), centrifuged and filtrate collected two times. The combined aqueous phases were concentrated in vacuo to yield the product [Cp*Ir(H2O)3]SO4 as a yellow solid (1.15 g, 94%).

Screening 1
The initial reaction conditions were based on previously published method. 3 The commercially available MozN3 was chosen as nitrogen source. Low solubility of the NaOAc and AgNTf2 additives was noted according to the conditions of entry 1. Highest conversion was observed according to conditions of entry 1. Conditions of entry 8 were chosen for further optimization, as lower reaction concentrations were sought after to facilitate the use of stock solutions.
Reaction scale 0.05 mmol. The reactions were set up in a 24-well Para-dox block, vials equipped with stirrer bars. The solid additives and catalysts were weighed in manually. N-(tert-butyl)benzamide (8.9 mg, 0.05 mmol) and MozN3 (15.5mg, 0.075 mmol) were added, followed by addition of the reaction solvent. The reaction mixtures were stirred at 500 rpm and heated at 60 °C for 20h. After this DMSO (200 µL) and metal scavenger were added to the vials and stirred for additional 2 hours at room temperature. Analyzed by LCMS.

MozN3 and catalyst loading effect study
The reactions were set up in a 96-well Para-dox gold block equipped with stirrer bars. Reaction scale 0.02 mmol. [Cp*Ir(H2O)3]SO4 (0.05M) was added as solutions in deionized water. The solvent was then removed using a Genevac HT6 centrifuge evaporator. The substrates were weighed in manually. N-(2-Pyrimidyl)indole (3.9 mg, 0.02 mmol) was added as solid using the Quantos powder dosing system. To the same vials MozN3 (0.3M, 100 µL, 0.03 mmol) was added in respective solvents. 2-(3methylphenyl)pyridine (0.02 mmol) and MozN3 (0.03 mmol) were added together as stock solution (100 µL per vial) in respective solvents. The reaction mixtures were stirred at 500 rpm and heated at 60 °C for 20h. After this DMSO (200 µL) and metal scavenger were added to the vials and stirred for additional 2 hours at room temperature. Analyzed by LCMS.

Directing group informer library
The reactions were set up in a 96-well Para-dox gold block equipped with stirrer bars. Reaction scale 0.02 mmol. [Cp*Ir(H2O)3]SO4 (0.05M, 40 µL, 2 µmol) was added as solutions in deionized water. The solvent was then removed using a Genevac HT6 centrifuge evaporator. The solid substrates were weighed in manually. After this, MozN3 (0.3M, 100 µL, 0.03 mmol) was added as stock solution in respective solvents. Addition of liquid substrates (0.02 mmol) followed. The reaction mixtures were stirred at 500 rpm and heated at 60 °C for 20h. After this DMSO (200 µL) and metal scavenger were added to the vials and stirred for additional 2 hours at room temperature. Analyzed by LCMS.

Successful examples, identity confirmed by NMR analysis at screening scale. Conversion determined by LCMS (UV trace).
Below: Purification was not attempted due to low conversion (top row) or insufficient separation in analytical LCMS (bottom row).

Conversion determined by LCMS (UV trace).
Below: Compounds with failed purification. The four examples presented are counted as unsuccessful reactions.

Below: Compounds with no conversion observed
Analyzed by LCMS (UV trace). Below: Substrate not chosen for purification due to low conversion or product decomposition. Counted as unsuccessful reactions.

Conversion determined by LCMS (UV trace). a Product with O-deacetylation under reaction conditions.
LSF Scope: side reaction

Conversion determined by LCMS (UV trace). Mirabegron analogue isolated and characterized.
In all depicted cases formation of a new species (% conversion in parentheses) was observed, in all cases with a MW lower by 15 Dalton then for the expected products. In the case of Mirabegron the product was isolated and confirmed as the addition product depicted. As the common feature of the compounds with which this mass difference was observed was the presence of highly nucleophilic functional groups, at this point we speculate that the same side reaction occurred in all presented cases.

Below: Compounds with failed purification
Conversion determined by LCMS (UV trace).Potential reasons for failed purification: Decomposition during sample preparation and/or purification, lack of ionization and detection of predicted product mass during the purification run, or observation of false positives by LCMS.

Miniaturization studies
Reaction scale 0.2 -0.8 µmol The reactions were set up in a 384-well Plate+™ glass-coated microplate. Stock solutions S1 were prepared with the respective substrates (0.256 mmol) and MozN3 (0.256 mmol) in 640 µL NMP. Stock solution S2 was prepared from [Cp*Ir(H2O)3]SO4 (0.04 mmol) in 1 mL NMP. Reactions were run at three distinct volumes: 1, 2 and 4 µL. A column control experiments with no catalyst was also set up for each substrate. The stock solutions were dispensed to the reaction plate using the Mosquito liquid dispensing system, with S1 added first, followed by S2. The plate was sealed and heated at 60 °C in a Genevac centrifuge at atmospheric pressure (Note: Using the centrifuge for the reaction minimized solvent loss). The plate was allowed to reach ambient temperature, centrifuged for 5 min at 2500 rpm, seal removed. Metal scavenger (Si-IMI, 2 equiv) was added in NMP. The reactions were diluted with NMP to a total of 40 µL. The plate was sealed and heated at 50 °C on an orbital shaker. An analytical plate was made, with 2 mM concentration, solvent DMSO. Analyzed by LCMS. Reactions were successful throughout the plate, including the lowest reaction volume (1 µL, see manuscript).

Reaction scale 1.0 -8.0 mmol
The reactions were set up in a 1536-well echo plate. Stock solutions S1 were prepared with the respective substrates (0.1 mmol) and MozN3 (0.1 mmol) in 233 µL NMP. Stock solution S2 was prepared from [Cp*Ir(H2O)3]SO4 (8 µmol) in 200 µL NMP. Reactions were run at four distinct volumes: 5, 10, 20 and 40 nL. The stock solutions were dispensed to the reaction plate using the Echo acoustic liquid handling system, with S1 added first, followed by S2. The plate was sealed and heated at 60 °C in a Genevac centrifuge at atmospheric pressure (Note: Using the centrifuge for the reaction minimized solvent loss). An analytical plate was made, with 2 mM concentration, solvent water. Analyzed by LCMS and AMI-MS. Product detection by MS was observed throughout the plate, even at the lowest reaction volumes (5 nL). 4-methoxybenzyl (3-methyl-2-(pyridin-2-yl)phenyl)carbamate (2c) was prepared according to General Procedure A. Purification by automated flash column chromatography (0-80% EtOAc in heptane, 25g SiO2). Compound 2c was obtained as a colorless solid (152.5 mg, 88%).